Adiponectin is an adipokine that we first identified in the mid-90's that has gained significant attention due to its insulin sensitizing and anti-inflammatory properties. Many papers have belabored the function of adiponectin in the widest spectrum of clinical and preclinical settings, yet there is no unifying mechanism of action available. Here, we would like to build upon our current data and develop further the mechanistic aspects of adiponectin action. We would like to approach this with the following questions: SA 1) What is the mechanism for the potent anti-apoptotic properties exerted by adiponectin on a number of cell types? We have generated a series of mouse models that allow us to provide an inducible pro-apoptotic insult to specific cell types. These ATTAC mice (Apoptosis Through Triggered Activation of Caspase-8) are available for pancreatic cells, cardiac myocytes, podocytes and several other critical cell types. We have preliminary genetic data highlighting the potent adiponectin-dose dependent effects on survival of cardiac myocytes in this model. We will extend these studies to cells and podocytes. The basis for these observations is a reduction of local ceramides and/or an increase in the local sphingosine-1-P levels due to activation of the adiponectin receptors by adiponectin. We will extend our preliminary data and believe that the ceramide-lowering effects of adiponectin mediated by the adiponectin receptors AdipoR1 and AdipoR2 offers the first unifying mechanism of action by which adiponectin achieves its remarkable effects systemically on a wide variety of cells. SA 2) Determine whether there is an intracrine mechanism of action for adiponectin within the adipocyte by which it enhances the metabolic flexibility of adipose tissue. Adiponectin overexpression from the liver is dramatically different from adiponectin overexpression from the adipocyte. In order to achieve the remarkable degree of metabolic flexibility that adipocyte-specific overexpression achieves, we postulate that there is an intracellular mechanism of action that ensures enhanced differentiation potential, increased glyceroneogenesis, improvements in vascularization and enhanced susceptibility to 3 adrenergic action. Using a combination of novel transgenic models and in vitro biochemistry we will put this hypothesis to a test. SA 3) Identify additional intracellular steps that lead to the assembly/maturation of adiponectin and effective release from adipocytes. We have identified Erp44 and Ero1 as critical factors for the assembly of adiponectin. We would like to identify additional critical players in this complex set of reactions, and establish the role of the Unfolded Protein Response (a process frequently upregulated in obese adipose tissue) in the maturation process. Combined, these studies should provide significant new insights into the physiology of this protein. The proposed lowering of ceramide levels is an attractive novel mechanism with the potential to explain the insulin-sensitizing, anti-apoptotic, pro-angiogenic and anti-inflammatory properties that have been attributed to adiponectin.